ジャーナル: PLoS One / 年: 2022 タイトル: Cryo-electron structures of the extreme thermostable enzymes Sulfur Oxygenase Reductase and Lumazine Synthase. 著者: Mohamed A Sobhy / Lingyun Zhao / Dalaver Anjum / Ali Behzad / Masateru Takahashi / Muhammad Tehseen / Alfredo De Biasio / Rachid Sougrat / Samir Hamdan / 要旨: Thermostable enzymes have the potential for use in a wide variety of biotechnological applications. Cryo-electron microscopy (cryo-EM) enables the imaging of biomolecules in their native aqueous ...Thermostable enzymes have the potential for use in a wide variety of biotechnological applications. Cryo-electron microscopy (cryo-EM) enables the imaging of biomolecules in their native aqueous environment. Here, we present high resolution cryo-EM structures of two thermostable enzymes that exhibit multimeric cage-like structures arranged into two different point-group symmetries. First, we determined the structure of the Sulfur Oxygenase Reductase (SOR) enzyme that catalyzes both the oxygenation and disproportionation of elemental sulfur in Archea and is composed of 24 homomeric units each of MW ≃ 35 kDa arranged in octahedral symmetry. The structure of SOR from Acidianus ambivalens (7X9W) was determined at 2.78 Å resolution. The active site of each subunit inside the central nanocompartment is composed of Fe3+ coordinated to two water molecules and the three amino acids (H86, H90 and E114). Second, we determined the structure of Lumazine Synthase (LS) from Aquifex aeolicus (7X7M) at 2.33 Å resolution. LS forms a cage-like structure consisting of 60 identical subunits each of MW ≃ 15 kDa arranged in a strict icosahedral symmetry. The LS subunits are interconnected by ion-pair network. Due to their thermostability and relatively easy purification scheme, both SOR and LS can serve as a model for the catalytic and structural characterization of biocatalysts as well as a benchmark for cryo-EM sample preparation, optimization of the acquisition parameters and 3D reconstruction.
装置: FEI VITROBOT MARK I / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 295 K 詳細: The purified protein was applied to the glow discharged grids inside the chamber, blotted for a duration of 1.5 s, and then plunge-frozen in liquid ethane cooled by liquid nitrogen.